HPD residues crucial for stimulation of HSP70 ATPase activity diverged in candidate orthologues of spoke HSP40. (A) Blastp in NCBI with human TSAR6 revealed a branch of an HSP40 phylogenic tree. The branch contained C. reinhardtii RSP16 (arrow), C. intestinalis spoke HSP40, and one TSAR6-like molecule from evolutionarily diverse organisms. Partial or redundant sequences were removed for clarity. (B) Multiple sequence alignment of DnaJ domains in TSAR6-like molecules from A demonstrated extensive sequence homology. However, the residues in the loop between the sequence corresponding to helixes II and III diverged significantly. Notably, the HPD tripeptides (asterisks) were only retained in the spoke HSP40 from C. reinhardtii, C. intestinalis, and sea urchin. H, D (arrowheads), and the two subsequent residues (dots) for stimulating HSP70 enzymatic activities diverged. The identical and similar residues are shaded in black and gray, respectively.